Global Transcriptomic Changes Research Articles

Abstract B012: HNRNPL-mediated mRNA stability underlies metastasis triggered by resistance to radiation therapy

Abstract The high rate of locoregional/distant recurrence in triple-negative breast cancer (TNBC) is a major driver of the decreased survival observed in breast cancer patients. Radiation therapy is a reliable treatment modality to limit recurrence for many cancers, but TNBC patients have a higher chance for relapse despite receiving increased doses of radiation compared to patients with other breast cancer subtypes. This study investigated the differential pathways in radioresistant TNBC tumors that impact recurrence. We developed two radioresistant cell line models (468-RR and 4T1-RR) by administering a radiation dose of 50Gy over the course of 8 weeks to the human TNBC cell line, MDA-MB-468, and the mouse TNBC cell line, 4T1, respectively. Migration and invasion were assessed using the scratch-wound assay and transwell assay, respectively. We assessed in vivo metastasis by injecting the cells in the mammary fat pad, tail vein, or left ventricle. RNA-sequencing of the cell lines was done to identify global transcriptomic changes and regulatory pathways induced by radiation therapy. The mRNA stability of integrin b3 was assessed by inhibiting transcription using actinomycin D. Moreover, we implemented the newly developed InduroRT-mediated circRNA-sequencing (IMRT-seq) to identify the circular RNAs processed by HNRNPL that are present in radioresistant cells. We observed enhanced lung and bone metastasis, based on bioluminescent imaging, in both the spontaneous and vascular metastasis models for the radioresistant cells compared to the parental cells. Genset enrichment analysis identified mRNA stability and metabolism as upregulated pathways in radioresistant cells with HNRNPL, a ribonucleoprotein involved in mRNA stability, as one of the top genes. By knocking down HNRNPL, we observed decreased migration capacity of the radioresistant cells. Given the role of HNRNPL in integrin and extracellular matrix genes, which are key components associated with metastasis in cancer cells, we screened for these genes in our RNA-seq dataset and identified integrin b3 as a possible target. More specifically, knocking down HNRNPL decreased the mRNA stability of integrin b3 as assessed by actinomycin d treatment. By expressing integrin b3 in the HNRNPL-depleted cells, we were able to rescue the migration capacity of these cells. We believe a subset of HNRNPL-mediated circular RNAs are responsible for integrin b3 mRNA stability by sequestering miRNAs that target integrin b3. With IMRT-seq we are exploring the potential circRNA-miRNA interactions present in radioresistant TNBC responsible for its metastatic phenotype. The radioresistant cells derived from TNBC cell lines have an enhanced metastatic potential than the treatment-naive cell lines and our data suggest that RNA metabolism is pivotal to this phenotype. The upregulation of HNRNPL upon radiation by Nrf2 increases integrin b3 expression by stabilizing its transcripts and preventing its degradation. This novel mechanism of radioresistant metastasis provides a therapeutic approach to minimize TNBC recurrence. Citation Format: Ayush Kumar, Kensei Kishimoto, Christi Wisniewski, Shivam Goel, Arthur Mercurio. HNRNPL-mediated mRNA stability underlies metastasis triggered by resistance to radiation therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B012.

Therapeutic potential of astrocyte-derived extracellular vesicles in mitigating cytotoxicity and transcriptome changes in human brain endothelial cells

This study investigates the therapeutic effect of astrocyte-derived extracellular vesicles (EVs) in mitigating neurotoxicity-induced transcriptome changes, mitochondrial function, and base excision repair mechanisms in human brain endothelial cells (HBECs). Neurodegenerative disorders are marked by inflammatory processes impacting the blood–brain barrier (BBB) that involve its main components- HBECs and astrocytes. Astrocytes maintain homeostasis through various mechanisms, including EV release. The effect of these EVs on mitigating neurotoxicity in HBECs has not been investigated. This study assesses the impact of astrocyte-derived EVs on global transcriptome changes, cell proliferation, cytotoxicity, oxidative DNA damage, and mitochondrial morphology in HBECs exposed to the neurotoxic reagent Na2Cr2O7. Exposure to Na2Cr2O7 for 5 and 16 h induced oxidative DNA damage, measured by an increase in genomic 8OHdG, while the EVs reduced the accumulation of the adduct. A neurotoxic environment caused a non-statistically significant upregulation of the DNA repair enzyme OGG1 while the addition of astrocyte-derived EVs was associated with the same level of expression. EVs caused increased cell proliferation and reduced cytotoxicity in Na2Cr2O7-treated cells. Mitochondrial dysfunction associated with a reduced copy number and circular morphology induced by neurotoxic exposure was not reversed by astrocyte-derived EVs. High-throughput RNA sequencing revealed that exposure to Na2Cr2O7 suppressed immune response genes. The addition of astrocyte-derived EVs resulted in the dysregulation of long noncoding RNAs impacting genes associated with brain development and angiogenesis. These findings reveal the positive impact of astrocytes-derived EVs in mitigating neurotoxicity and as potential therapeutic avenues for neurodegenerative diseases.

CMTM6 mediates the Warburg effect and promotes the liver metastasis of colorectal cancer

Liver metastasis of colorectal cancer (CRC) is a leading cause of death among cancer patients. The overexpression of glucose transporter 1 (Glut1) and enhanced glucose uptake that are associated with the Warburg effect are frequently observed in CRC liver metastases, but the underlying mechanisms remain poorly understood. CKLF-like MARVEL transmembrane domain-containing protein 6 (CMTM6) regulates the intracellular trafficking of programmed death-ligand-1 (PD-L1); therefore, we investigated whether CMTM6 regulates Glut1 trafficking and the Warburg effect in CRC cells. We found that knocking down of CMTM6 by shRNA induced the lysosomal degradation of Glut1, decreased glucose uptake and glycolysis in CRC cells, and suppressed subcutaneous CRC growth in nude mice and liver metastasis in C57BL/6 mice. Mechanistically, CMTM6 forms a complex with Glut1 and Rab11 in the endosomes of CRC cells, and this complex is required for the Rab11-dependent transport of Glut1 to the plasma membrane and for the protection of Glut1 from lysosomal degradation. Multiomics revealed global transcriptomic changes in CMTM6-knockdown CRC cells that affected the transcriptomes of adjacent cancer-associated fibroblasts from CRC liver metastases. As a result of these transcriptomic changes, CMTM6-knockdown CRC cells exhibited a defect in the G2-to-M phase transition, reduced secretion of 60 cytokines/chemokines, and inability to recruit cancer-associated fibroblasts to support an immunosuppressive CRC liver metastasis microenvironment. Analysis of TCGA data confirmed that CMTM6 expression was increased in CRC patients and that elevated CMTM6 expression was associated with worse patient survival. Together, our data suggest that CMTM6 plays multiple roles in regulating the Warburg effect, transcriptome, and liver metastasis of CRC.

High-throughput transcriptomics toxicity assessment of eleven data-poor bisphenol A alternatives

Bisphenol A (BPA), a widely used chemical in the production of plastics and epoxy resins, has garnered significant attention due to its association with adverse health effects, particularly its endocrine-disrupting properties. Regulatory measures aimed at reducing human exposure to BPA have led to a proliferation of alternative chemicals used in various consumer and industrial products. While these alternatives serve to reduce BPA exposure, concerns have arisen regarding their safety and potential toxicity as regrettable substitutes. Previous efforts have demonstrated that in vitro high-throughput transcriptomics (HTTr) studies can be used to assess the endocrine-disrupting potential of BPA alternatives, and this strategy produces transcriptomic points-of-departure (tPODs) that are protective of human health when compared to the PODs from traditional rodent studies. In this study, we used in vitro HTTr to assess the potential for toxicity of eleven data-poor legacy chemicals sharing structural similarities to BPA. Human breast cancer MCF-7 cells were exposed to BPA and 11 alternatives at concentrations ranging from 0.1 to 25 μM to assess toxicity. Analysis of global transcriptomic changes and a previously characterized estrogen receptor alpha (ERα) transcriptomic biomarker signature revealed that 9 of 11 chemicals altered gene expression relative to controls. One of the chemicals (2,4′-Bisphenol A) activated the ERα biomarker at the same concentration as BPA (i.e., 4,4′-BPA) but was deemed to be more potent as it induced global transcriptomic changes at lower concentrations. These results address data gaps in support of ongoing screening assessments to identify BPA alternatives with hazard potential and help to identify potential candidates that may serve as safer alternatives.

Transcriptome and proteome changes triggered by overexpression of the transcriptional regulator Maf1 in the human pathogen Leishmania major.

Maf1, originally described as a repressor of RNA polymerase III (RNAP III) transcription in yeast, participates in multiple functions across eukaryotes. However, the knowledge about Maf1 in protozoan parasites is scarce. To initiate the study of Maf1 in Leishmania major, we generated a cell line that overexpresses this protein. Overexpression of Maf1 led to a significant reduction in the abundance of tRNAs, 5S rRNA, and U4 snRNA, demonstrating that Maf1 regulates RNAP III activity in L. major. To further explore the roles played by Maf1 in this microorganism, global transcriptomic and proteomic changes due to Maf1 overexpression were determined using RNA-sequencing and label-free quantitative mass spectrometry. Compared to wild-type cells, differential expression was observed for 1082 transcripts (615 down-regulated and 467 up-regulated) and 205 proteins (132 down-regulated and 73 up-regulated) in the overexpressing cells. A correlation of 44% was found between transcriptomic and proteomic results. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the differentially expressed genes and proteins are mainly involved in transcription, cell cycle regulation, lipid metabolism and transport, ribosomal biogenesis, carbohydrate metabolism, autophagy, and cytoskeleton modification. Thus, our results suggest the involvement of Maf1 in the regulation of all these processes in L. major, as reported in other species, indicating that the functions performed by Maf1 were established early in eukaryotic evolution. Notably, our data also suggest the participation of L. major Maf1 in mRNA post-transcriptional control, a role that, to the best of our knowledge, has not been described in other organisms.

Abstract Mo073: Elucidating the Role of Cardiac Radiotherapy on the Sympathetic Nervous System as a Treatment for Ventricular Tachycardia

Ventricular tachycardia (VT) is a dangerous arrhythmia that occurs in structurally and electrically heterogenous regions of the diseased heart, leading to sudden cardiac arrest and death if left untreated. One of the main drivers underlying the arrhythmogenic substrate is abnormal remodeling of the autonomic nervous system after cardiac injury, resulting in a heterogenous distribution of sympathetic innervation and subsequent ventricular arrhythmia. In patients with VT refractory to conventional therapy, noninvasive cardiac stereotactic body radiotherapy (SBRT) has emerged as a promising new treatment. Here, we investigated whether cardiac radiotherapy homogenizes the arrhythmogenic substrate through induction of cardiac sympathetic nerve innervation and activity in a closed-chest ischemia reperfusion murine model of ischemic heart failure. Using single nucleus-RNA sequencing, we identified global transcriptomic changes in the left ventricle 6 weeks after 25 Gray (Gy) cardiac X-ray radiation. Notably, these changes included increased expression of neurotrophins such as brain derived neurotrophic factor, a growth factor essential for neuronal survival and development, in irradiated hearts compared to sham controls. Consequently, Western blot and immunofluorescence showed increased expression of the sympathetic neuronal markers tyrosine hydroxylase and neuropeptide Y at the scar border zone of irradiated mice. Similar immunofluorescence results were observed at the irradiated border zone from the explanted heart of a patient previously targeted with 25 Gy compared to a nontargeted remote region within the same heart. These findings suggest a novel role for radiation-induced remodeling of the sympathetic nervous system in the anti-arrhythmic response to SBRT.

Transcriptomic Change in the Effects of Dichloroquinolinic Acid on the Development and Growth of Nicotiana tabacum

Dichloroquinolinic acid is a hormone-type herbicide widely used to control barnyard grass during crop cultivation. However, it can seriously inhibit the growth of susceptible crops, including tobacco, because it degrades slowly under field conditions. Additionally, the mechanism by which it damages crops is unclear. More specifically, the transcriptional changes in plants induced by dichloroquinolinic acid remain unknown. In this study, differentially expressed genes (DEGs) in tobacco treated with dichloroquinolinic acid (varying concentrations and durations) were analyzed and validated to explore the global transcriptome changes. The number of DEGs, which were determined according to the FPKM, varied from 758 to 21,340. The KEGG analysis revealed that many DEGs were involved in starch and sucrose metabolism, phenylpropanoid biosynthesis, photosynthesis, porphyrin and chlorophyll metabolism, and glutathione metabolism. Transcriptomic analyses indicated that dichloroquinolinic acid can inhibit tobacco growth by inhibiting photosynthesis and storage of energy. We discovered that the toxicity mechanism of the hormone herbicide dichloroquinolinic acid differs from that of high concentrations of IAA (Indoleacetic acid), despite studies confirming that the effects of hormone herbicides are consistent with the physiological disturbances and growth inhibition exhibited by plants in IAA overdose. Particularly, dichloroquinolinic acid suppresses photosynthesis while high concentration IAA stimulates nucleotide synthesis and photosynthesis. More importantly, we found by editing the IAA-responsive gene IAA16, tobacco could develop resistance to dichloroquinolinic acid. The results will help clarify plant responses to hormone-type herbicides at the transcriptional level, thereby providing insights into the diversity in the gene’s response to herbicides, the molecular targets of hormone-type herbicides, and the mechanism underlying the susceptibility of tobacco to dichloroquinolinic acid. Accordingly, this study may be helpful for future research to enhance crop resistance to herbicides residues.

ATRT-14. TARGETING ATYPICAL TERATOID RHABDOID TUMORS USING A NOVEL BI-FUNCTIONAL COREST INHIBITOR

Abstract BACKGROUND Atypical teratoid/rhabdoid tumors (ATRT) are deadly infantile brain tumors driven by a loss-of-function mutation of SMARCB1, a critical component of the SWI-SNF chromatin remodeling complex. However, residual SWI-SNF activity inhibits EZH2 at all loci except where CoREST colocalizes with these complexes, specifically contributing to ATRT pathogenesis. This results in hypermethylation and subsequent repression of genes coding for neuronal differentiation and tumor suppressors, accounting for ATRTs’ stem-like state, aggressive growth, and therapy resistance. We functionally inhibited the CoREST complex in ATRT cells by targeting its core enzymes LSD1 and HDAC1/2 using a bi-functional inhibitor, corin. METHODS We evaluated the efficacy of corin to target ATRT cells (BT37, CHLA05 and CHLA06) by measuring cell proliferation (BrdU immunofluorescence/IF), apoptosis (CC3 IF, cPARP Western blotting/WB, MUSE Annexin V), histone modifications (WB) and neuronal differentiation (WB, IF). Whole mRNA sequencing and ATAC-Seq were performed to see the global transcriptomic and chromatin changes driven by corin. Further, we examined the long-term survival advantage rendered by corin in an in-vivo orthotopic xenograft model of ATRT. RESULTS Corin reprogrammed the chromatin structure in ATRT cells in favor of pathways related to neuronal differentiation, cell differentiation, and axonal development while downregulating pathways related to proliferation and cell cycle (mRNA Seq, ATAC-Seq). Corin inhibited AT/RT cell growth and proliferation (p<0.05), increased apoptosis (p<0.05), increased H3K4me1 and H3K9Ac levels (p < 0.001), and induced neuronal differentiation (IF: Beta-3-tubulin, MAP2, Synaptophysin). Intratumoral delivery of corin increased the median survival of mice bearing aggressive orthotopic CHLA06 tumors from 14 to 35 days compared to control (p=0.02, Log Rank test). CONCLUSIONS Targeting the CoREST complex with corin disrupts ATRT’s epigenetic abnormalities, reversing their stem-like state and slowing tumor growth, thus presenting a promising new strategy that may translate into the clinical setting to help improve ATRT’s dismal survival.

Assessing Transcriptomic Responses to Oxidative Stress: Contrasting Wild-Type Arabidopsis Seedlings with dss1(I) and dss1(V) Gene Knockout Mutants.

Oxidative stress represents a critical facet of the array of abiotic stresses affecting crop growth and yield. In this paper, we investigated the potential differences in the functions of two highly homologous Arabidopsis DSS1 proteins in terms of maintaining genome integrity and response to oxidative stress. In the context of homologous recombination (HR), it was shown that overexpressing AtDSS1(I) using a functional complementation test increases the resistance of the Δdss1 mutant of Ustilago maydis to genotoxic agents. This indicates its conserved role in DNA repair via HR. To investigate the global transcriptome changes occurring in dss1 plant mutant lines, gene expression analysis was conducted using Illumina RNA sequencing technology. Individual RNA libraries were constructed from three total RNA samples isolated from dss1(I), dss1(V), and wild-type (WT) plants under hydrogen peroxide-induced stress. RNA-Seq data analysis and real-time PCR identification revealed major changes in gene expression between mutant lines and WT, while the dss1(I) and dss1(V) mutant lines exhibited analogous transcription profiles. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed significantly enriched metabolic pathways. Notably, genes associated with HR were upregulated in dss1 mutants compared to the WT. Otherwise, genes of the metabolic pathway responsible for the synthesis of secondary metabolites were downregulated in both dss1 mutant lines. These findings highlight the importance of understanding the molecular mechanisms of plant responses to oxidative stress.

Cryptococcus neoformans Slu7 ensures nuclear positioning during mitotic progression through RNA splicing.

The position of the nucleus before it divides during mitosis is variable in different budding yeasts. Studies in the pathogenic intron-rich fungus Cryptococcus neoformans reveal that the nucleus moves entirely into the daughter bud before its division. Here, we report functions of a zinc finger motif containing spliceosome protein C. neoformans Slu7 (CnSlu7) in cell cycle progression. The budding yeast and fission yeast homologs of Slu7 have predominant roles for intron 3' splice site definition during pre-mRNA splicing. Using a conditional knockdown strategy, we show CnSlu7 is an essential factor for viability and is required for efficient cell cycle progression with major role during mitosis. Aberrant nuclear migration, including improper positioning of the nucleus as well as the spindle, were frequently observed in cells depleted of CnSlu7. However, cell cycle delays observed due to Slu7 depletion did not activate the Mad2-dependent spindle assembly checkpoint (SAC). Mining of the global transcriptome changes in the Slu7 knockdown strain identified downregulation of transcripts encoding several cell cycle regulators and cytoskeletal factors for nuclear migration, and the splicing of specific introns of these genes was CnSlu7 dependent. To test the importance of splicing activity of CnSlu7 on nuclear migration, we complemented Slu7 knockdown cells with an intron less PAC1 minigene and demonstrated that the nuclear migration defects were significantly rescued. These findings show that CnSlu7 regulates the functions of diverse cell cycle regulators and cytoskeletal components, ensuring timely cell cycle transitions and nuclear division during mitosis.

O16 Understanding the pleiotropic effects of the Rho-kinase inhibitor Y-27632 on human keratinocytes

Abstract Introduction and aims Successful epidermal transplant generation depends on sufficiently high numbers of self-renewing stem cells that give rise to clonal colonies termed ‘holoclones’. Treatment with Y-27632, a nonspecific Rho-kinase 1/2 inhibitor, conditionally immortalizes epidermal keratinocyte cultures. However, the pleiotropic signalling mechanisms downstream of Y-27632 remain largely unexplored. We have therefore characterized the effects of continuous long-term Y-27632 treatment on chromatin regulation, and investigated whether Y-27632 reprogrammes a holoclone-like state. Methods Neonatal foreskin-derived human epidermal keratinocytes were cultured from passage p5 to p30, with or without Y treatment. Samples were collected for RNA sequencing analysis from progenitor-enriched middle passage cells (p5, MPK), progenitor-depleted late-passage cells (p8, LPK) and Y-treated cells at p8 (LPK-Y). Samples were also collected for assay for transposase-accessible chromatin (ATAC)-Seq analysis to assess open and closed chromatin regions. Results Untreated cultures became depleted of proliferating cells by p11, whereas Y maintained a highly proliferative culture up to p30 and beyond. Y withdrawal at p11 resulted in rapid loss of proliferative capacity within a single passage. ATAC-Seq identified 196 differentially regulated chromatin sites in MPK vs. LPK. Y treatment maintained accessibility in approximately 40% of these MPK-enriched chromatin regions. Approximately 3000 differentially expressed genes (DEGs) were identified in MPK vs. LPK, while Y treatment induced approximately 11 000 DEGs. Activated genes in LPK-Y were enriched for a holoclone signature, with no evidence for activation of a precancerous transcriptome. Y treatment altered the expression of various genes associated with amino acid metabolism, enabling cells to overcome serine/glycine autotrophy. Conclusions Y acts by preserving accessibility of progenitor-enriched chromatin sites and further reprogrammes the keratinocyte transcriptome towards a more holoclone-like state. However, keratinocytes retain a ‘memory’ of their doubling number, which is conditionally suppressed by Y. The observed global transcriptomic changes are indicative of the highly pleiotropic nature of the Y-induced signalling changes, warranting further deconvolution.

Telomerase is essential for cardiac differentiation and sustained metabolism of human cardiomyocytes

Telomeres as the protective ends of linear chromosomes, are synthesized by the enzyme telomerase (TERT). Critically short telomeres essentially contribute to aging-related diseases and are associated with a broad spectrum of disorders known as telomeropathies. In cardiomyocytes, telomere length is strongly correlated with cardiomyopathies but it remains ambiguous whether short telomeres are the cause or the result of the disease. In this study, we employed an inducible CRISPRi human induced pluripotent stem cell (hiPSC) line to silence TERT expression enabling the generation of hiPSCs and hiPSC-derived cardiomyocytes with long and short telomeres. Reduced telomerase activity and shorter telomere lengths of hiPSCs induced global transcriptomic changes associated with cardiac developmental pathways. Consequently, the differentiation potential towards cardiomyocytes was strongly impaired and single cell RNA sequencing revealed a shift towards a more smooth muscle cell like identity in the cells with the shortest telomeres. Poor cardiomyocyte function and increased sensitivity to stress directly correlated with the extent of telomere shortening. Collectively our data demonstrates a TERT dependent cardiomyogenic differentiation defect, highlighting the CRISPRi TERT hiPSCs model as a powerful platform to study the mechanisms and consequences of short telomeres in the heart and also in the context of telomeropathies.

Abstract 1662: Evaluation of TEAD inhibitor, VT03989 treatment on growth of aggressive meningioma preclinical models

Abstract Background: Aggressive meningiomas (WHO grade II/III) are incurable brain tumors and despite surgery and radiation therapy, tumors recur frequently without approved systemic treatment options. Hence, there is an unmet need for targeted inhibition therapy and biomarkers that predict vulnerability to targeted therapies in aggressive meningioma patients. Hippo signaling pathway has been shown to be dysregulated in meningioma, resulting in activation of its target TEAD as a transcriptional factor. We hypothesize that targeted inhibition of TEAD in recurrent high-grade meningioma with NF2 loss will lead to increased inhibition of tumor growth. Here, we investigated the effect of VT3989, a potent TEAD inhibitor, in meningioma cell lines with NF2 wild-type or NF2 loss genotype. Methods: Two meningioma cell-lines: IOMM-Lee (NF2 wild-type) and MG309, a grade III meningioma patient-derived cell line with NF2 loss were treated with various doses of VT3989 and cell viability was assessed. Relative expression of direct targets of TEAD including CTGF and CYR61 was determined with quantitative PCR (qPCR) to ensure target engagement. Global transcriptomic changes were examined via RNAseq analysis on RNA isolated from MG309 cells treated with VT03989 compared to vehicle treated cells. Results: VT3989 treatment significantly decreased viability of both IOMM-Lee and MG309 cells. Canonical downstream targets of TEAD such as CTGF, CYR61, BIRC5, BCL-2 were downregulated in VT3989 treated cells compared to control. Furthermore, VT03989 treatment resulted in downregulation in cell cycle regulation and DNA damage response (DDR) pathway genes. Ongoing work is focused on combining VT3989 with agents targeting cell cycle and DDR pathways. Conclusion: VT3989 treatment led to downregulation of TEAD direct targets CTGF and CYR61 and inhibited cell viability of both IOMM-Lee cell and MG309 meningioma lines. RNA-seq analysis identified cell cycle and DDR pathway genes to be downregulated upon TEAD inhibition. Citation Format: Siddhi Desai, Anil Thaghalli Shivanna, Mariya Stavnichuk, Elizabeth Hayes, Sonam Patel, James McNamara, Anita DeSantis, Nader Sanai, An-Chi Tien, Shwetal Mehta. Evaluation of TEAD inhibitor, VT03989 treatment on growth of aggressive meningioma preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1662.

Comparative Transcriptome Analysis of Babesia bigemina Attenuated Vaccine and Virulent Strains of Mexican Origin.

Bovine babesiosis, caused by the protozoan Babesia bigemina, is one of the most important hemoparasite diseases of cattle in Mexico and the world. An attenuated B. bigemina strain maintained under in vitro culture conditions has been used as a live attenuated vaccine; however, the biological mechanisms involved in attenuation are unknown. The objective of this study was to identify, through a comparative transcriptomics approach, the components of the B. bigemina virulent parasites that are differentially expressed in vivo, as opposed to those expressed by B. bigemina attenuated vaccine parasites when inoculated into naïve cattle. The biological material under study was obtained by inoculating spleen-intact cattle with infected erythrocytes containing either the attenuated strain or a virulent field strain. After RNA extraction, transcriptomic analysis (RNA-seq) was performed, followed by bioinformatic Differential Expression (DE) analysis and Gene Ontology (GO) term enrichment. The high-throughput sequencing results obtained by analyzing three biological replicates for each parasite strain ranged from 9,504,000 to 9,656,000, and 13,400,000 to 15,750,000 reads for the B. bigemina attenuated and virulent strains, respectively. At least 519 differentially expressed genes were identified in the analyzed strains. In addition, GO analysis revealed both similarities and differences across the three categories: cellular components, biological processes, and molecular functions. The attenuated strain of B. bigemina derived from in vitro culture presents global transcriptomic changes when compared to the virulent strain. Moreover, the obtained data provide insights into the potential molecular mechanisms associated with the attenuation or pathogenicity of each analyzed strain, offering molecular markers that might be associated with virulence or potential vaccine candidates.

Stable knockdown of Drp1 improves retinoic acid-BDNF-induced neuronal differentiation through global transcriptomic changes and results in reduced phosphorylation of ERK1/2 independently of DUSP1 and 6.

Background: Dynamin-related protein Drp1 -a major mitochondrial fission protein- is widely distributed in the central nervous system and plays a crucial role in regulating mitochondrial dynamics, specifically mitochondrial fission and the organelle's shaping. Upregulated Drp1 function may contribute to the pathological progression of neurodegenerative diseases by dysregulating mitochondrial fission/ fusion. The study aims to investigate the effects of Drp1 on retinoic acid-BDNF-induced (RA-BDNF) neuronal differentiation and mitochondrial network reorganization in SH-SY5Y neuroblastoma cells. Methods: We generated an SH-SY5Y cell line with stably depleted Drp1 (shDrp1). We applied RNA sequencing and analysis to study changes in gene expression upon stable Drp1 knockdown. We visualized the mitochondria by transmission electron microscopy and used high-content confocal imaging to characterize and analyze cell morphology changes and mitochondrial network reorganization during neuronal differentiation. Results: shDrp1 cells exhibited fused mitochondrial ultrastructure with perinuclear clustering. Stable knockdown of Drp1 resulted in the upregulation of genes involved in nervous system development. High content analysis showed improved neurite outgrowth, segmentation, and extremities in differentiated shDrp1 cells. Neuronal differentiation was associated with a significant reduction in ERK1/2 phosphorylation, and ERK1/2 phosphorylation was independent of the dual specificity phosphatases DUSP1/6 in shDrp1 cells. Differentiated control underwent mitochondrial morphology remodeling, whereas differentiated shDrp1 cells retained the highly fused mitochondria and developed long, elongated structures. The shDrp1 cells responded to specific apoptotic stimuli like control in vitro, suggesting that Drp1 is not a prerequisite for apoptosis in SH-SY5Y cells. Moreover, Drp1 downregulation reduced the formation of toxic mHtt aggregates in vitro. Discussion: Our results indicate that Drp1 silencing enhances RA-BDNF-induced neuronal differentiation by promoting transcriptional and mitochondrial network changes in undifferentiated cells. We also demonstrate that the suppression of Drp1 reduces toxic mHtt aggregate formation in vitro, suggesting protection against neurotoxicity. Thus, Drp1 may be an attractive target for further investigation in future strategies to combat neurodegenerative diseases.

Chronic and acute exposure to rotenone reveals distinct Parkinson's disease-related phenotypes in human iPSC-derived peripheral neurons

Peripheral autonomic nervous system (P-ANS) dysfunction is a critical non-motor phenotype of Parkinson's disease (PD). The majority of PD cases are sporadic and lack identified PD-associated genes involved. Epidemiological and animal model studies suggest an association with pesticides and other environmental toxins. However, the cellular mechanisms underlying toxin induced P-ANS dysfunctions remain unclear. Here, we mapped the global transcriptome changes in human induced pluripotent stem cell (iPSC) derived P-ANS sympathetic neurons during inhibition of the mitochondrial respiratory chain by the PD-related pesticide, rotenone. We revealed distinct transcriptome profiles between acute and chronic exposure to rotenone. In the acute stage, there was a down regulation of specific cation channel genes, known to mediate electrophysiological activity, while in the chronic stage, the human P-ANS neurons exhibited dysregulation of anti-apoptotic and Golgi apparatus-related pathways. Moreover, we identified the sodium voltage-gated channel subunit SCN3A/Nav1.3 as a potential biomarker in human P-ANS neurons associated with PD. Our analysis of the rotenone-altered coding and non-coding transcriptome of human P-ANS neurons may thus provide insight into the pathological signaling events in the sympathetic neurons during PD progression.

The identification of a Distinct Astrocyte Subtype that Diminishes in Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by the presence of two hallmark pathologies: the accumulation of Amyloid beta (Aβ) and tau proteins in the brain. There is a growing body of evidence suggesting that astrocytes, a type of glial cell in the brain, play crucial roles in clearing Aβ and binding to tau proteins. However, due to the heterogeneity of astrocytes, the specific roles of different astrocyte subpopulations in response to Aβ and tau remain unclear. To enhance the understanding of astrocyte subpopulations in AD, we investigated astrocyte lineage cells based on single-nuclei transcriptomic data obtained from both human and mouse samples. We characterized the diversity of astrocytes and identified global and subpopulation-specific transcriptomic changes between control and AD samples. Our findings revealed the existence of a specific astrocyte subpopulation marked by low levels of GFAP and the presence of AQP4 and CD63 expression, which showed functional enrichment in Aβ clearance and tau protein binding, and diminished in AD. We verified this type of astrocytes in mouse models and in AD patient brain samples. Furthermore, our research also unveiled significant alterations of the ligand-receptor interactions between astrocytes and other cell types. These changes underscore the complex interplay between astrocytes and neighboring cells in the context of AD. Overall, our work gives insights into astrocyte heterogeneity in the context of AD and reveals a distinct astrocyte subpopulation that holds potential for therapeutic interventions in AD. Targeting specific astrocyte subpopulations may offer new avenues for the development of novel treatments for AD.

ZmELP1, an Elongator complex subunit, is required for the maintenance of histone acetylation and RNA Pol II phosphorylation in maize kernels.

The Elongator complex was originally identified as an interactor of hyperphosphorylated RNA polymerase II (RNAPII) in yeast and has histone acetyltransferase (HAT) activity. However, the genome-wide regulatory roles of Elongator on transcriptional elongation and histone acetylation remain unclear. We characterized a maize miniature seed mutant, mn7 and map-based cloning revealed that Mn7 encodes one of the subunits of the Elongator complex, ZmELP1. ZmELP1 deficiency causes marked reductions in the kernel size and weight. Molecular analyses showed that ZmELP1 interacts with ZmELP3, which is required for H3K14 acetylation (H3K14ac), and Elongator complex subunits interact with RNA polymerase II (RNAPII) C-terminal domain (CTD). Genome-wide analyses indicated that loss of ZmELP1 leads to a significant decrease in the deposition of H3K14ac and the CTD of phosphorylated RNAPII on Ser2 (Ser2P). These chromatin changes positively correlate with global transcriptomic changes. ZmELP1 mutation alters the expression of genes involved in transcriptional regulation and kernel development. We also showed that the decrease of Ser2P depends on the deposition of Elongator complex-mediated H3K14ac. Taken together, our results reveal an important role of ZmELP1 in the H3K14ac-dependent transcriptional elongation, which is critical for kernel development.

Blockade of histone deacetylase activity affects transcription and splicing of neuronal and glial genes

The study of the molecular mechanisms underlying plastic processes in the nervous system is of great interest in modern neuroscience. It is important to understand that epigenetic modifications, which are crucial for the development and cellular differentiation, can also be involved in plastic processes in the adult nervous system. In our early work, we provided evidence that the expression of important memory-related genes, such as Prkcz and Prkci, can be regulated epigenetically [1]. In the current study, we extended the previous work to the systemic level by applying the RNA sequencing approach to evaluate global changes in the expression patterns of various genes during the induction of epigenetic rearrangements. Rat cortical neuron cultures were incubated with one of the nonselective histone deacetylase inhibitors (trichostatin A, TSA; sodium butyrate, NaB) to change the level of epigenetic regulation. Next, the total RNA was extracted and subjected to RNA-Seq libraries preparation and subsequent NGS-sequencing. Bioinformatics analysis of transcriptomic data revealed substantial overlapping of differentially expressed genes (DEGs) in NaB-treated and TSA-treated groups, indicating that different histone deacetylase (HDAC) inhibitors induce transcriptional changes in primary neuron cultures through common regulatory pathways irrespective of chemical structure of applied inhibitor. We found that histone deacetylase blockade is accompanied by a transition from proliferative processes to cellular differentiation. Gene Ontology (GO) analysis of DEGs datasets revealed that the upregulated genes were engaged in cell differentiation and specialization, tissue and embryonic morphogenesis, and the development of various peripheral tissues and organs. On the contrary, genes that reduce the expression under induction of epigenetic rearrangements were involved in biological processes associated with cell proliferation and, most interestingly, the specialization of various brain cells (neurons, astrocytes, oligodendrocytes). It was shown that the expression of a number of glial markers typical for astrocytes and oligodendrocytes was significantly reduced after application of HDAC inhibitors, which was also confirmed by quantitative PCR using specific primer pairs on selected target genes. During the data analysis, we also found a significant decrease in the expression of various neuronal markers associated with the cytoskeleton, the organization of pre- and postsynaptic endings, synaptic transmission. It is known that fine-tuning of various processes in the central nervous system is due to the production of different isoforms of proteins from the same gene due to the process of alternative splicing of the resulting mRNA. According to the literature, epigenetic rearrangements create a certain environment for the regulation of alternative splicing of different genes [2, 3]. It is shown that production of alternative isoforms can play an important role in various plastic processes [4, 5]. Therefore, using IsoformSwitchAnalyzeR and DEXSeq packages we analyzed the possibility of alternative splicing during the induction of epigenetic rearrangements in rat cortical neuron cultures by evaluating the abundance of various transcripts based on exon usage. We found that some glial genes and a large number of neuronal genes, especially those associated with postsynaptic organization and cell communication, were alternatively spliced after application of histone deacetylase inhibitors. Inhibition of HDAC activity in cortical neuron cultures mainly affected the choice of alternative transcription starts (ATSS) and terminators (ATTS), and to a lesser extent alternative splicing of exons. Obtained results were selectively confirmed by the quantitative PCR using specific pairs of primers for individual exons of different transcript isoforms. Thus, within this study, it was found that histone deacetylases play an important role in the specialization of various brain cells, and the suppression of their activity affects the expression and alternative splicing of various glial and neuronal marker genes. We do not exclude that global transcriptome changes caused by alternative splicing will lead to qualitative rearrangements of the neuron network, and this is the direction of future research.

The BET degrader ZBC260 suppresses stemness and tumorigenesis and promotes differentiation in triple-negative breast cancer by disrupting inflammatory signaling

BackgroundBreast cancer stem cells (BCSCs) are resistant to standard therapies, facilitate tumor dissemination, and contribute to relapse and progression. Super-enhancers are regulators of stemness, and BET proteins, which are critical for super-enhancer function, are a potential therapeutic target. Here, we investigated the effects of BET proteins on the regulation of breast cancer stemness using the pan-BET degrader ZBC260.MethodsWe evaluated the effect of ZBC260 on CSCs in TNBC cell lines. We assessed the effect of ZBC260 on cellular viability and tumor growth and measured its effects on cancer stemness. We used RNA sequencing and stemness index to determine the global transcriptomic changes in CSCs and bulk cells and further validated our findings by qPCR, western blot, and ELISA.ResultsZBC260 potently inhibited TNBC growth both in vitro and in vivo. ZBC260 reduced stemness as measured by cell surface marker expression, ALDH activity, tumorsphere number, and stemness index while increasing differentiated cells. GSEA analysis indicated preferential downregulation of stemness-associated and inflammatory genes by ZBC260 in ALDH+ CSCs.ConclusionsThe BET degrader ZBC260 is an efficient degrader of BET proteins that suppresses tumor progression and decreases CSCs through the downregulation of inflammatory genes and pathways. Our findings support the further development of BET degraders alone and in combination with other therapeutics as CSC targeting agents.